Luteoloside attenuates renal Ischemia–Reperfusion injury by suppressing ferroptosis through disruption of the KEAP1–NRF2 interaction

BACKGROUND: Renal ischemia-reperfusion (I/R) injury represents a principal etiological factor in acute kidney injury (AKI), with ferroptosis emerging as a key pathogenic mechanism. Luteoloside (Lut), a natural flavonoid with antioxidative properties, has not yet been well characterized in renal I/R injury. PURPOSE: The study explored the renoprotective effects of Lut and its underlying mechanisms. METHODS: We established renal I/R in C57BL/6 mice and hypoxia/reoxygenation (H/R)-induced injury in HK-2 cells. Transcriptomic analysis was used to investigate Lut-regulated pathways in renal I/R-related pathways. H&E staining, renal function assays, and apoptosis assay were performed to evaluate the protective effect of Lut, whereas ROS assay, MDA assay, iron content assay, and scanning electron microscopy were performed to assess ferroptosis-associated changes. To clarify the mechanism of Lut, molecular docking, CETSA, co-immunoprecipitation (Co-IP), and ubiquitination assays were used to examine Lut-KEAP1 interactions and the regulation of NRF2. RESULTS: Lut pretreatment significantly improved renal function after I/R, as evidenced by reduced serum creatinine and blood urea nitrogen levels, and markedly attenuated tubular injury and apoptosis in vivo. Lut reduced reactive oxygen species accumulation, malondialdehyde levels, and labile iron content, while restoring glutathione levels and rescuing GPX4 and SLC7A11 expression that was suppressed by I/R in mouse kidneys and HK-2 cells. Transcriptomic profiling revealed significant enrichment of antioxidant and ferroptosis-related pathways, with prominent activation of NRF2 signaling. Mechanistically, Lut directly bound to KEAP1, disrupted the KEAP1-NRF2 interaction, inhibited NRF2 ubiquitination, and promoted NRF2 nuclear translocation. Genetic silencing or pharmacological inhibition of NRF2 markedly attenuated the antioxidant, anti-ferroptotic, and renoprotective effects of Lut. CONCLUSION: This study demonstrates that Lut protects against renal I/R injury by suppressing ferroptosis through direct modulation of the KEAP1-NRF2 axis, supporting Lut as a potential redox-targeted therapy for I/R-induced AKI.